Islet beta-cell failure is central to the development of type 2 diabetes and is contributed to by both hyperglycemia and hyperlipidemia. The protein neprilysin, a component ofthe renin-angiotensin system (RAS), has been shown in non-islet tissues to be upregulated with the chronically elevated glucose and fat levels seen in type 2 diabetes. While the function of neprilysin in islets has not been investigated, we have data that show it is synthesized and active in islets and may play a role in the modulation of beta-cell function in response to islet stressors. Thus, the overall aim of this proposal is to elucidate the role of neprilysin under conditions associated with impaired beta-cell function, namely increased fat and glucose. We hypothesize that in states of chronically elevated fat and glucose, neprilysin activity is upregulated thereby promoting beta-cell dysfunction. During the K99 phase of this award, studies were conducted to determine the contribution of neprilysin to impaired insulin secretion induced by chronically elevated fat. Our findings demonstrate that neprilysin is indeed upregulated under conditions of chronically elevated fat and this is associated with reduced glucosestimulated insulin secretion in vitro. Moreover, neprilysin deficient (NEP-KO) mice fed a high fat diet are protected from high fat diet-induced reductions in glucose-stimulated insulin secretion in vivo. During the ROO phase of this award, the following studies will be performed to determine: 1) The role of neprilysin in the impairment Of insulin secretion induced by chronically elevated glucose. Our preliminary data suggest that neprilysin may also be upregulated under high glucose conditions and thereby may mediate the induction of oxidative stress. Firstly, NEP-KO mice will receive a 48-hour glucose infusion to induce hyperglycemia, then insulin secretion wil! be measured in vivo. Secondly, isolated islets from NEPKO mice will be cultured in high glucose and insulin secretion will be examined in vitro. 2) The role ofthe RAS in mediating neprilysin's effects under chronically elevated fat and glucose conditions. Since neprilysin is a component of the RAS, isolated islets will be used to determine whether neprilysin's activity in this pathway is responsible for impaired beta-cell function.